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P-A5-11 Synthesis and characterization of artificial proteins with random sequences
A5 Design of proteins and protein ligands P-A5-10 CALCULATIONS OF PROTONATION EQUILIBRIA FOR NATIVE AND COMPLEXED FORMS OF HIV-1 PROTEASE TRYLSKA J., ANTOSIEWICZ J. & GELLER M.
P-A5-09 The association of 24p3 protein with lipophilic ligands Yee-HsiungChen’,*,Sin-TakChu*, Han-Jia Lin’ and Kuang Ming Nien’ ‘In&Weof Biochemical Sciences. NationalTaiwan University and*Instituteof Biological Chemistry, Academia Sinica, Taipei,Taiwan
Dept. of Biophys.
& ICM, University
of Warsaw
(PL).
Purpose: The knowledge of protonotion equilibria of ionizablegroups in proteins is essential for understandingcatalytic mechanismsand interactionsbetween enzymesand their inhibitors, Methods: The calculationsof protein pKa’swere basedon the Poisson-Boltzmannmodel which describeselectrostaticinteractions. Results: The analysiswas focusedon two catalytic aspartatesin the binding site (Asp 25 and Asp 125). In the native proteasethere is no signiticant preferencefor any specificprotonation state. Binding of the inhibitor (e.g. MVT-101) shifts the protonation equilibria towards one predominant state. The protonation statesof the inhibitors are also affected. The effective molecularelectrostatic potential was computed for se&ted protonation states. Conclusions: Detailed analysisof the protonation equilibria is essentialfor further molecular modeling and in particularfor rational drug design. For more information see:Antosiewicz et al., J. Comp. Chem., 1996, in press. Geller,M. et al., Proteins.Structure,Function and Genetics,1996, in press.
The N-terminus of P glycopmtein derived frmn 24~3 mRNA was determined to be pymglutamate. Thii enabled us to elucidate the positions of the hvo hypmphans. namely Trp” and Trp”, atong the putative mature protein from the 24~3 d)NA-deduced protein squetbx. Tit& envimmnenu were stndied by hbinsic fluonsance and salute quenching. They give no emission peak at 332 mn and about 20.8% of them are accessible to queocbmg by acrylamide. Correlating tbc quencbiug effect of CsCl and IU on the protein fluorescence to the charge groups along the polypeptide chain suggests the difference in the “local charge” around the two tryptopban residues. Addition of each lipophilic ligaod such as fatty acid, retinoids and chole.sk%yl oleate to the protein solution effects a considemble chaoge in the pmfde of circular dichmism arising from the chromophores of protein in 200 - 300 mn end diminishes the tt-yptophan fluorescence. Ooe the other hand. cholesterol alone shows uo effect on tie protein fluorescence. Analysis of the fluorescence dam suggests that the protetu has a lipophilic site with the association constant of 1.03 X 10’ M”. 1.92 X ld ML. 2.38 X 10’ M” or 1.25 X ld M’ for chole.steryi oleate. oleate, rctinol or retinoic acid at pH 7.4. Analysis of the cquilibrimn binding data from binding assay using [H’]-retinol and [H’]-retiuoic acid reveals that the protein contains single class of retinoid-binding site with the association constant of 4.92 X 10’ ML and 1.17 X 10’ M’ for retinol and retinoic acid, respectively. The maximum binding capacity was determined to be 5.87 nmole win01 or 1.91 nmole retinoic acid I mg protein.
P-A>1 1 SYNTHESIS AND CHARACTERIZkTION OF ARTIFICIAL PROTEINS WITH RANDOM SEQUENCES Yomo T, PrijambadaID, Urabe I Depart.of Biotech.,OsakaUniv.(JAPAN)
P-A5-12
The investigation of synthetic proteins beyond the influence of natural evolution has come to light for knowing the general physicochemical properties of polypeptides. It gives us the basis for designing new protein structures that has not evolved in nature. A library of artificial proteins of 140 amino acid residues of which 95 is random, that includes 20 kinds of amino acids, was prepared Out of the 25 identified proteins, 5 are soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. The identified nucleotide sequences of the genes encoding the five soluble proteins and five arbitrarily chosen insoluble ones showed that statistically, the soluble proteins are less hydrophobic. In addition, the CD spectra and esterase activities of the purified RP3-42 and RP3-45, 2 out of the 5 soluble proteins, were also determined. From the gathered data, we proposed that a random polypeptide can be artificially evolved into an enzyme.
Lactobaciilus
Purpose: It is known that the Asn casei thydylate
TS) present altered substrate catalyzes refercntialIy the m versus d6 MP [Liu, L., & Biochem. 31, 5100-5104). consideration,a thermodynamicBon of the interaction betw~ dCMP and both wild-type and mutant TS has bam
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P-A5-09 The association of 24p3 protein with lipophilic ligands Yee-HsiungChen’,*,Sin-TakChu*, Han-Jia Lin’ and Kuang Ming Nien’ ‘In&Weof Biochemical Sciences. NationalTaiwan University and*Instituteof Biological Chemistry, Academia Sinica, Taipei,Taiwan
Dept. of Biophys.
& ICM, University
of Warsaw
(PL).
Purpose: The knowledge of protonotion equilibria of ionizablegroups in proteins is essential for understandingcatalytic mechanismsand interactionsbetween enzymesand their inhibitors, Methods: The calculationsof protein pKa’swere basedon the Poisson-Boltzmannmodel which describeselectrostaticinteractions. Results: The analysiswas focusedon two catalytic aspartatesin the binding site (Asp 25 and Asp 125). In the native proteasethere is no signiticant preferencefor any specificprotonation state. Binding of the inhibitor (e.g. MVT-101) shifts the protonation equilibria towards one predominant state. The protonation statesof the inhibitors are also affected. The effective molecularelectrostatic potential was computed for se&ted protonation states. Conclusions: Detailed analysisof the protonation equilibria is essentialfor further molecular modeling and in particularfor rational drug design. For more information see:Antosiewicz et al., J. Comp. Chem., 1996, in press. Geller,M. et al., Proteins.Structure,Function and Genetics,1996, in press.
The N-terminus of P glycopmtein derived frmn 24~3 mRNA was determined to be pymglutamate. Thii enabled us to elucidate the positions of the hvo hypmphans. namely Trp” and Trp”, atong the putative mature protein from the 24~3 d)NA-deduced protein squetbx. Tit& envimmnenu were stndied by hbinsic fluonsance and salute quenching. They give no emission peak at 332 mn and about 20.8% of them are accessible to queocbmg by acrylamide. Correlating tbc quencbiug effect of CsCl and IU on the protein fluorescence to the charge groups along the polypeptide chain suggests the difference in the “local charge” around the two tryptopban residues. Addition of each lipophilic ligaod such as fatty acid, retinoids and chole.sk%yl oleate to the protein solution effects a considemble chaoge in the pmfde of circular dichmism arising from the chromophores of protein in 200 - 300 mn end diminishes the tt-yptophan fluorescence. Ooe the other hand. cholesterol alone shows uo effect on tie protein fluorescence. Analysis of the fluorescence dam suggests that the protetu has a lipophilic site with the association constant of 1.03 X 10’ M”. 1.92 X ld ML. 2.38 X 10’ M” or 1.25 X ld M’ for chole.steryi oleate. oleate, rctinol or retinoic acid at pH 7.4. Analysis of the cquilibrimn binding data from binding assay using [H’]-retinol and [H’]-retiuoic acid reveals that the protein contains single class of retinoid-binding site with the association constant of 4.92 X 10’ ML and 1.17 X 10’ M’ for retinol and retinoic acid, respectively. The maximum binding capacity was determined to be 5.87 nmole win01 or 1.91 nmole retinoic acid I mg protein.
P-A>1 1 SYNTHESIS AND CHARACTERIZkTION OF ARTIFICIAL PROTEINS WITH RANDOM SEQUENCES Yomo T, PrijambadaID, Urabe I Depart.of Biotech.,OsakaUniv.(JAPAN)
P-A5-12
The investigation of synthetic proteins beyond the influence of natural evolution has come to light for knowing the general physicochemical properties of polypeptides. It gives us the basis for designing new protein structures that has not evolved in nature. A library of artificial proteins of 140 amino acid residues of which 95 is random, that includes 20 kinds of amino acids, was prepared Out of the 25 identified proteins, 5 are soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. The identified nucleotide sequences of the genes encoding the five soluble proteins and five arbitrarily chosen insoluble ones showed that statistically, the soluble proteins are less hydrophobic. In addition, the CD spectra and esterase activities of the purified RP3-42 and RP3-45, 2 out of the 5 soluble proteins, were also determined. From the gathered data, we proposed that a random polypeptide can be artificially evolved into an enzyme.
Lactobaciilus
Purpose: It is known that the Asn casei thydylate
TS) present altered substrate catalyzes refercntialIy the m versus d6 MP [Liu, L., & Biochem. 31, 5100-5104). consideration,a thermodynamicBon of the interaction betw~ dCMP and both wild-type and mutant TS has bam
66